Project2:MacrophageLipidHomeostasisandInflammatorySignaling ABSTRACT/SUMMARY The objective of Project 2 of this PPG is to understand how cellular lipid composition and lipid trafficking influence the inflammatory function of macrophages. Although perturbations in lipid homeostasis are recognized to be associated with inflammation in a number of human diseases, our understanding of ?how? and ?why? remains limited. Recent work has revealed that pro-inflammatory signals reprogram the lipid metabolic state of macrophages. It has also become clear that perturbations in lipid homeostasis can be sensed by the inflammatory machinery of macrophages so as to induce and to regulate inflammatory responses.Thus,lipidhomeostasisandinflammationareinterrelated,andperturbationsinoneaffecttheother. In this project, our PPG team will combine advanced analytical mass spectrometry?based approaches with genetic models of inflammation, with the goal of defining mechanisms by which inflammation drives reprogrammingofthelipidome(andviceversa).Wewillassesstheconsequencesofchangingthesubcellular levelsoflipidsoninflammatorysignaling.SpecificAim1istoapplyadvancedanalytictechniquestodetermine how pro- and anti-inflammatory signals change the subcellular lipidome in macrophages. We will use mass spectrometry approaches, including shotgun lipidomics, NanoSIMS imaging, and isotope labeling, to understanding how pro- and anti-inflammatory signals influence lipid localization and trafficking in macrophages.SpecificAim2istodeterminethemechanismsbywhichalterationsincholesterolhomeostasis potentiatetheSTINGsignalingpathway.Wewillpursueourdiscoverythatperturbationsindenovocholesterol synthesis change type I IFN inflammatory responses via STING. Using a combination of biochemical approaches, confocal and NanoSIMS imaging, and chemoproteomics, we will test the hypothesis that cholesterol regulates STING function through direct binding. We will also test whether disease-associated mutations in STING abrogate the regulatory impact of cholesterol. Specific Aim 3 is to determine the importance of the STING signaling pathway on the development of dyslipidemia, inflammation, and atherogenesisinmice.TypeIIFNshavebeenshowntoinfluencethepathogenesisofatherosclerosis,butthe molecular pathways underlying this sterile inflammatory response have not been elucidated. We will test the hypothesis that the cGAS/STING inflammatory axis is required to generate type I IFN in the setting of dyslipidemia and atherosclerosis. These studies will define the influence of the STING pathway on dyslipidemia, inflammation, immune cell infiltration, and atheroma development. It is our expectation thatour proposed studies will define, at a molecular level, why dysregulation of macrophage lipid homeostasis drives inflammation,andhowinflammationinfluencesmacrophagecholesterolmetabolismincardiovasculardisease. Our PPG team is excited by our hypotheses, and we are positioned, with all of the experimental approaches, reagents,andexpertcollaborators,tomakerapidprogress.